Freeze Tolerance

By Janet M. Storey

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A much more complex strategy for winter survival is freeze tolerance, the ability to endure the actual formation of ice within the body. Ice forming in body tissues can do a lot of harm. Ice crystals can puncture small blood vessels, squeeze and deform cells to the point of breaking, and scramble the micro- architecture inside cells so that upon thawing, organs are severely damaged. And even if ice doesn't break into cells, it leads to their severe dehydration, for water is sucked out of cells into the growing ice crystals leaving behind a shrunken and damaged cell. Freezing of the blood also interrupts the delivery of oxygen and nutrients to organs and so also causes severe metabolic damage. Any animal species that adopts a freeze tolerance strategy for winter survival must, therefore, find ways to overcome all of these types of injuries that can be caused by ice. Few, if any, organisms can deal with horrendous structural damage that is caused by ice formation inside of cells, so freeze tolerant animals typically confine ice growth to extracellular spaces of their bodies while using protective mechanisms to keep the water inside their cells from freezing (extracellular freeze tolerance plus intracellular freeze avoidance).

Ice crystals - They can be beautiful, but inside bodies they are deadly!

One of the most amazing examples of freeze tolerance is the wood frog (Rana sylvatica). This common frog is found across Canada and all the way north to the tree line. Wood frogs winter on land, hiding under the leaf litter on the forest floor. By hibernating on land, the frogs can get active as soon as the snow melts and breed in the temporary ponds and ditches formed by meltwater. Wood frog choruses can often be heard over a month before aquatic frogs, like leopard frogs, are released from their ice-locked lakes and rivers to begin their own breeding. But hibernation on land has its cost. Frogs can't dig underground like toads do and they need to hibernate in damp leaf litter to keep from drying out. Their soft, water-permeable skin is no barrier to ice and so, whenever frost penetrates into their winter home, they freeze. Ice penetrates though all of the fluid compartments of the animal and within just a few hours a mass of ice fills the abdominal cavity encasing all the internal organs. Large flat ice crystals run between the layers of skin and muscle, and the eyes turn white because the lens freezes. Their blood stops flowing and as much as 65% of the frog's total body water is converted to ice. Breathing, heart beat, and muscle movements all stop and the frozen "frog-sicle" exists in a virtual state of suspended animation until it thaws.

Gray Treefrog (Hyla versicolor) - The phrase: "So cold, you turn blue!" gains new meaning in the case of this frog whose skin does turn blue when it freezes! In Manitoba there are 5 species of frogs that winter on land and experience freezing: wood frog, boreal chorus frog, spring peeper, gray tree frog and Cope's gray tree frog.

How do they freeze and thaw without injury? Firstly, wood frogs and other freeze tolerant animals take active control over freezing. Rather than wait for spontaneous ice formation to begin, they employ special ice nucleators that actively seed ice formation in their bodies. In this way the animals can start freezing just below 0·C. This is helpful because the higher the freezing temperature, the slower the rate of ice formation and the longer the time available for the animal to make metabolic adjustments that ensure survival. Sometimes nucleators are bacteria on the skin or in the gut and sometimes they are special ice nucleating proteins that are added to blood.

Painted Turtle hatchlings - Baby painted turtles (Chrysemys picta) remain in the nest, just below the ground surface, for their first winter and experience freezing. They are the "highest" known vertebrate animal that freezes!

The second part of freezing survival is the same as that for freeze avoidance - the animals build up high concentrations of sugars or sugar alcohols in their tissues. In this case, however, the sugars are used to keep just the insides of cells from freezing, rather than the whole organism. So ice forms all around the outsides of the internal organs, sucking water out of them, but leaving behind a thick syrupy solution inside cells which can't freeze.

Many of the adaptations in frogs that support freezing survival grew out of pre-existing mechanisms that were first evolved by amphibians to deal with water stress. Because of their highly water-permeable skins, all terrestrial and semi-aquatic amphibians can experience wide daily variations in body water content and many frogs can recover after the loss of >50% of their total body water, an ability unparalleled among other vertebrates, including humans. To a frog cell there is no functional difference between the loss of water into the external environment versus into a mass of abdominal ice and the adaptations that regulate cell volume under one type of water stress work just as well for the other.

Most freeze tolerant insects use the sugar alcohol, glycerol, for antifreeze as do their freeze avoiding insect cousins, but the wood frog, and other frogs, use glucose which is the normal blood sugar of all vertebrate animals. What is unique for wood frogs, however, is that they easily tolerate blood sugar levels that are 100-fold or higher than normal and show none of massive injuries that are suffered by human diabetics when their blood sugar rises by only 2-10 fold. Hence, these frogs may have some important lessons to tell us about how an animal can manage hugely high sugar levels in its tissues without ill effect.

The third part to freeze tolerance is the need for all organs to survive through the freeze without any deliveries of oxygen or nutrients via the blood, which is frozen. The low body temperature during freezing helps, for metabolic rate is also very low, but all freeze tolerant animals have also enhanced the abilities of their organs to survive without oxygen.

Another aspect to freeze tolerance which has not yet been explored is the fact that, while frozen, organ functions cease - heart beat stops, breathing halts. It will be very interesting to find out what signals shut off these functions when body ice content rises to a certain level and how they are spontaneously reactivated after thawing. Finally, freeze tolerant animals also appear to enhance their body's damage repair mechanisms in order to deal with any physical injuries to organs that may be caused by ice while they are frozen. In wood frogs, for example, the levels of clotting proteins rise in the blood so that any bleeding that is detected during thawing can be quickly halted.

So, never think of winter as a boring season of dormancy and hibernation for the animal kingdom. Far from it. Every animal has evolved amazing adaptations to cope with life in the freezer and ensure a successful reawakening in the spring.

Thanks for learning about being Frozen Alive! Bye for now!

Biography

Jan Storey (M.Sc.) and her husband Ken (Ph.D., F.R.S.C., Professor of Biochemistry) are members of the Institute of Biochemistry at Carleton University in Ottawa. Their research over 20 years has explored the marvellous diversity of animal adaptation using the tools of biochemists to determine how changes in intermediary metabolism, protein and enzyme structure/function, and gene expression empower animals to adapt to environmental extremes. Much of their research centres on animal adaptation for life in the cold including studies of freeze tolerance and freeze avoidance, mammalian hibernation, and anoxia survival. You can contact the Storey's at: Janet Storey or Ken Storey.

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